Development and validation of a panel of five proteins as blood biomarkers for early detection of colorectal cancer

Figures & data

Figure 1 STARD diagram showing the selection of study participants enrolled in the BLITZ study during 2005–2015 and the analysis scheme.

Abbreviations: CRC, colorectal cancer; STARD, STAandards for the Reporting of Diagnostic accuracy studies.

Table 1 Characteristics of the study population in the discovery set and validation set

Group	Discovery set		Validation set
	CRC (N, %)	Control (N, %)	CRC (N, %)	Advanced adenoma (N, %)	Control (N, %)
Total	226	118	41	106	107
Age (years)
<60	50 (23.1)	49 (42.6)	7 (19.4)	37 (36.3)	46 (43.8)
60–64	32 (14.8)	29 (25.2)	11 (30.6)	31 (30.4)	22 (21.0)
65–69	30 (13.9)	17 (14.8)	5 (13.9)	10 (9.8)	17 (16.2)
≥70	104 (48.1)	20 (17.4)	13 (36.1)	24 (23.5)	20 (19.0)
Mean ± SD	67.8±12.0	62.0±7.3	66.8±6.9	63.5±7.1	62.2±6.3
Sex
Male	129 (57.1)	52 (44.1)	29 (70.7)	56 (52.8)	44 (41.1)
Female	97 (42.9)	66 (55.9)	12 (29.3)	50 (47.2)	62 (57.9)
UICC TNM tumor stage
I	67 (29.6)	–	14 (34.1)	–	–
II	64 (28.3)	–	3 (7.3)	–	–
III	67 (29.6)	–	21 (51.2)	–	–
IV	28 (12.4)	–	3 (7.3)	–	–
CRC location
Colon	139 (61.5)	–	23 (56.1)	–	–
Rectum	87 (38.5)	–	17 (41.5)	–	–
Unknown	–	–	1 (2.4)	–	–
Advanced adenoma subclass
HGD	–	–	–	14 (13.2)	–
Villous	–	–	–	56 (34.0)	–
Adenoma ≥ 1 cm	–	–	–	36 (52.8)	–

Abbreviations: CRC, colorectal cancer; HGD, high-grade dysplasia; UICC TNM, Union for International Cancer Control tumor node metastasis.

Table 2 Diagnostic performance of 12 significant protein markers

Protein marker	Discovery set		Validation set
	Fold change (CRC vs free of neoplasm)	p-value*	Fold change (CRC vs free of neoplasm)	p-value*
GDF-15	1.68	<0.001	1.42	<0.001
AREG	1.34	<0.001	1.32	<0.001
TRAILR-2	1.23	<0.001	1.28	<0.001
IL-6	1.56	<0.001	1.48	0.014
AM	1.23	<0.001	1.28	0.004
HE4	1.24	<0.001	1.19	0.014
TNFR-2	1.21	<0.001	1.16	0.014
ILT3	1.21	<0.001	1.14	0.043
CEA	1.29	<0.001	1.86	<0.001
CXCL9	1.27	0.001	1.42	0.040
TNFR-1	1.17	0.001	1.24	0.014
HGF	1.14	0.007	1.27	0.032

Note:

* p-values were adjusted for multiple testing (Benjamini–Hochberg method).

Abbreviations: AM, adrenomedullin; AREG, amphiregulin; CEA, carcinoembryonic antigen; CRC, colorectal cancer; CXCL9, C-X-C motif chemokine 9; GDF-15, growth differentiation factor 15; HGF, hepatocyte growth factor; HE4, WAP four-disulfide core domain protein 2; IL-6, interleukin-6; ILT3, Immunoglobulin-like transcript 3; TNFR-1, tumor necrosis factor receptor-1; TNFR-2, tumor necrosis factor receptor-2; TRAILR-2, TNF-related apoptosis-inducing ligand receptor-2.

Figure 2 Comparison of ROC curves of the four- and five-marker panel for detecting: (A) CRC vs controls free of neoplasm; (B) advanced adenomas vs controls free of neoplasm.

Abbreviations: AUC, area under the curve; CRC, colorectal cancer; ROC, receiver operating characteristic.

Table 3 Diagnostic performance of multi-marker panels for detecting CRC in the validation set

Marker panel	AUC (95% CI)	Sensitivity (95% CI)
		At 90% specificity	At 80% specificity
CRC vs controls free of neoplasm
Four-protein panel	0.81 (0.73–0.88)	53.6 (26.8–70.7)	63.4 (48.8–82.9)
Four-protein panel + TP53 autoantibody	0.82 (0.74–0.90)	56.4 (38.4–71.8)	66.7 (48.7–82.1)
Advanced adenomas vs controls free of neoplasm
Four-protein panel	0.58 (0.51–0.65)	18.9 (8.5–27.4)	23.6 (15.1–38.7)
Four -protein panel + TP53 autoantibody	0.60 (0.52–0.69)	22.0 (13.4–35.4)	31.7 (18.3–45.1)

Note: Four-protein panel includes GDF-15, AREG, Fas and Flt3L.

Abbreviations: AREG, amphiregulin; AUC, area under the curve; CRC, colorectal cancer; Flt3L, Fms-related tyrosine kinase 3 ligand; GDF-15, growth differentiation factor 15.

Figure 3 Comparison of ROC curves of (A) the four-marker panel and (B) five-marker panel for detecting early- and late-stage CRCs.

Abbreviations: AUC, area under the curve; CRC, colorectal cancer; ROC, receiver operating characteristic.